Diabetes mellitus is a metabolic disorder that results from insulin deficiency and is reflected by blood glucose concentrations being outside the normal range of 80-120 mg/dL (4.4-6.6 mM) [Ann. Intern. Med. 2007, 146, ITC1-15]. Diabetes causes complications such as neuropathy, nephropathy and retinopathy which result in heart disease, kidney failure, or blindness, respectively [Klonoff D C. Noninvasive blood glucose monitoring, Diabetes Care 1997; 20: 433-437]. Therefore, in order to treat diabetes, it is very important for diabetics to control their blood glucose levels by conducting self-monitoring several times a day. Indeed, glucose biosensors account for about 85% of the entire biosensor market. Such a huge demand in the market makes diabetes a model disease for developing new biosensing concepts.
A wide variety of methods for glucose analysis, including electrochemistry, near infrared spectroscopy, optical rotation and the like, have been reported in the literature [Yokowama, K., Sode, K., Tamiya, E., Karube, I. Anal. Chim. Acta (1989), 218, 137; Rabinovitch, B., March, W. F., Adams, R. L. Diabetes Care (1982), 5, 254; G. M., Moses, R. G., Gan, I. E. T., Blair, S. C. Diabetes Res. Clin. Pract. (1988), 4, 177; D_Auria, S., Dicesare, N., Gryczynski, Z., Gryczynski, I.; Rossi, M.; Lakowicz, J. R. Biochem. Biophys. Res. Commun. (2000), 274, 727]. The most commonly used technology for blood glucose determination is an enzyme-based method.
Electrochemical glucose monitoring has greatly contributed to improving the lives of diabetic patients. Despite the impressive progress in the development of electrochemical glucose biosensors, there are still many challenges and obstacles related to the achievement of a highly stable, enzyme-free and reliable glucose monitoring devices. The development in the last five decades is summarized in a recent review [J. Wang, Chem. Rev. 2008, 108, 814˜825].
In general, the detection of glucose by electrochemical biosensors is based on the electrochemical oxidation of hydrogen peroxide generated by enzyme-catalyzed oxidation of glucose at anodic potentials (>+0.6 V vs. Ag/AgCl) [J. Wang, N. Naser, L. Anges, W. Hui, L. Chen, Anal. Chem. 64 (1992) 1285-1288]. However, at this relatively high potential, there may be interferences from other oxidizable species such as ascorbic acid, uric acid and acetaminophen. The glucose oxidase (GOx) based-glucose devices rely on the use of oxygen as the physiological electron acceptor, and they are subject to errors resulting from fluctuations in the oxygen pressure and the stoichiometric limitation of oxygen. Few strategies have been evolved to circumvent the oxygen deficiency [Wang, J.; Mo, J. W.; Li, S. F.; Porter, J. Anal. Chim. Acta (2001), 441, 183; D'Costa, E.; Higgins, I., Turner, A. P. Biosensors (1986), 2, 71]. Also, innovative methodologies have been adapted for establishing and tailoring the electrical contact between the redox center of GOx and electrode surfaces to improve the electron transport [Pishko, M. V., Katakis, I., Lindquist, S. E., Ye, L., Gregg, B. A., Heller, A. Angew. Chem., Int. Ed. (1990), 29, 82; Riklin, A., Katz, E., Willner, I., Stocker, A., Buckmann, A. F. Nature (1995), 376, 672].
Recently, research studies have been focused on eliminating the mediator and developing a reagentless glucose biosensor with a low operating potential close to the redox potential of the enzyme. In this case, the electron is transferred directly from glucose to the electrode via the active site of the enzyme. The absence of mediators is the main advantage of such third-generation biosensors and results in a very high selectivity (owing to the very low operating potential). The development in nanotechnology has inspired the application of nanomaterials in bioanalytical chemistry.
For the fabrication of a high-efficiency biosensor, the selection of a substrate matrix for dispersing the sensing material decides the sensor performance. It is highly desirable to use the substrates that high-surface area, optimum porosity, high thermal stability, chemical inertness and minimum or negligible swelling in aqueous and non-aqueous solutions. Electrospun fibrous membranes meet many of the requirements for achieving improved performance for a sensor electrode. The chief advantages of electrospun fibrous materials include design flexibility, dimensional stability upon the flow of gases and liquids through fiber bundles, high-surface area, safer operations, ease of scaling up, and reusability. Electrospun nanofibrous materials high surface area-to-volume ratios which are suitable for improving biosensor characteristics. Biological molecules can be immobilized onto the surface of electrospun membranes. However, the molecules on the surface of electrospun fibers tend to leach out when the fibrous mat is placed in a solution. It is therefore important to minimize the leaching of the biomolecules/enzymes within the fibrous mat using an additional functional material that can bind the biological molecules/enzymes.
Glucose was assayed amperometrically by the GOx-electroreduction of a ferricinium cation to a ferrocene, which was then electrooxidized on the screen printed carbon-paste electrode of a strip [Kyvik, K. O., Traulsen, J., Reinholdt, B., Froland, A. Diabetes Res. Clin. Pract. (1990), 10, 8590]. Home blood-glucose monitors utilize plastic or paper strips comprising electrochemical cells and contain PQQGDH, NAD-GDH, FAD-GDH or GOx and a redox mediator. These glucose monitors can utilize amperometry or chronoamperometry or coulometry.
The enzyme-based glucose sensors have a lot of problems in terms of the stability of the enzyme, oxygen dependence, the role of the mediator, enzyme leaching, etc. GOx quickly loses its activity below pH 2 and above pH 8, and is rapidly deteriorated at a temperature higher than 40° C. (R. Wilson, A. P. F. Turner, Biosens. Bioelectron. 7 (1992) 165]. Relatively high or low humidity may adversely affect the storage and use of the sensors. Due to these problems, the development of an enzymeless glucose sensor is required.
For the development of a practical non-enzymatic glucose sensor, suitable electrocatalysts have been used. Platinum surfaces modified by a heavy metal such as Tl, Pb, Bi, or WO3 exhibited catalytic activity for glucose oxidation [G. Kokkinidis, N. Xonoglou, Bioelectrochem. Bioenerg. 14 (1985) 375; G. Wittstock, A. Strubing, R. Szargan, G. Werner, J. Electroanal. Chem. 444 (1998) 61; X. Zhang, K.-Y. Chan, J.-K. You, Z.-G. Lin, A. C. C. Tseung, J. Electroanal. Chem. 430 (1997) 147]. However, the catalytic oxidation has been limited to acidic or basic conditions. Non-enzymatic glucose sensors have been fabricated using nanoporous platinum [S. Park, T. D. Chung, H. C. Kim, Anal. Chem., (2003), 75, 3046; H. Boo, S. Park, B. Ku, Y. Kim, J. H. Park, H. C. Kim, T. D. Chung, J. Am. Chem. Soc., (2004), 126, 4524]. Most of the non-enzymatic glucose sensors, which have been suggested to date, have no glucose-recognition unit.
Recently, the present inventors have demonstrated the utilities of a non-enzymatic glucose sensor based on an electrospun nanoporous functional membrane [K. M. Manesh, P. Santhosh, A. Gopalan, Kwang-Pill Lee, Analytical Biochemistry, 2007, 360, 189]. A novel sensor electrode based on a composite electrospun nanofiberous membrane of poly(vinylidene fluoride) (PVdF) and poly(aminophenylboronic acid) (PAPBA) was fabricated on an indium tin oxide (ITO)-coated glass plate. The glucose-sensing ability of the nanofibrous membrane was assessed and, as a result, the PVdF/PAPBA-NFM exhibited an excellent linear response to the detection of glucose in the concentration range of 1 to 15 mM within a response time of less than 6 seconds. The PVdF/PAPBANFM fabricated through an electrospinning process enabled glucose to be detected with high selectivity and sensitivity even in the presence of other carbohydrates and showed negligible interference, reproducibility, and storage stability. The excellent performance of the nanofibrous membrane is attributable to its lager surface area and active sites suitable for glucose sensing. The electrospun membrane-based glucose sensor is ideal for glucose sensing in flowing streams. However, the process of fabricating the biosensor by depositing the electrospun PVdF/PAPBA-NFM directly on the electrode surface (ITO) has a problem in that it is difficult to control the thickness and uniformity of the surface.